Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery tothe liver

We are developing a self-assembling non-viral in vivo gene delivery vehicle based on poly-L-lysine and plasmid DNA, We have characterized poly-L-lysin es of different chain lengths for DNA condensation and strength of DNA bind ing. Poly-L-lysine chains > 20 residues bound DNA efficiently in physiologi cal saline, while shorter chains did not. Attachment of asialoorosomucoid t o PLL increased the PLL chain length required for efficient DNA binding in saline and for efficient DNA condensation. By electron microscopy, poly-L-l ysine/DNA polyplexes appeared as toroids 25-50 nm in diameter or rods 40-80 nm long; conjugation of asialoorosomucoid to the polylysine component incr eased the size of resulting polyplexes to 50-90 nmn. In water, poly-L-lysin e and asialoorosomucoid-PLL polyplexes have effective diameters of 46 and 8 7.6 nm, respectively. Polyplexes containing only poly-L-lysine and DNA aggr egated in physiological saline at all charge ratios and aggregated at neutr al charge ratios in water. Attachment of asialoorosomucoid lessened, but di d not eliminate, the aggregation of PLL polyplexes, and did not result in e fficient delivery of polyplexes to hepatocytes. Conjugation of polyethylene glycol to poly-L-lysine sterically stabilized resulting polyplexes at neut ral charge ratios by shielding the surfaces. For efficient in vivo gene del ivery, polyplexes will need to be sterically stabilized to prevent aggregat ion and interaction with serum components. (C) 1999 Published by Elsevier S cience B.V. All rights reserved.